第五章从基因文库中克隆基因.ppt

玉林师范学院化生系 Packaging the cosmid recombinants into phage coats imposes a desirable selection upon their size. With a cosmid vector of 5 kb, we demand the insertion of 32-47 kb of foreign DNA - much more than a phage-λ, vector can accommodate. Note that, after packaging in vitro, the particle is used to infect a suitable host. The recombinant cosmid DNA is injected and circularizes like phage DNA but replicates as normal plasmid without the expression of any phage functions. Transformed cells are selected on the basis of a vector drug-resistance marker. Cosmids provide all efficient means of cloning large pieces of foreign DNA. Even with sized foreign DNA in practice cosmid clones may be produced that contain non-contiguous DNA fragments ligated to form a single insert. The problem can be solved by dephosphorylating（去磷酸） the foreign DNA fragments so as to prevent their ligation together. This method is very sensitive to the exact ratio of target-to-vector DNAs because vector-to-vector ligation can occur. Furthermore, recombinants with a duplicated vector are unstable and break down in the host by recombination, resulting in the propagation of a non-recombinant cosmid vector. How to solve this problem? Such difficulties have been overcome in a cosmid-cloning procedure devised by Ish-Horowicz and Burke . By appropriate treatment of the cosmid vector pJB8 (Fig. 5.2) , left-hand and right-hand vector ends are purified which are incapable of self ligation but which accept dephosphorylated(脱磷酸) foreign DNA. Thus the method eliminates the need to size the foreign DNA fragments and prevents formation of clones containing short foreign DNA or multiple vector sequences. Fig. 5.2 Cosmid cloning scheme of Ish-Horowicz and Burke (1981 ). (a) Map of cosmid pJB8. (b) Application to the construction of a genomic library of fragments obtained by partial digestion with suu3A. This restriction endonuclease has a tetranucleotide(四核苷酸) recognition site and generation fragments with